Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG

a novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8. This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents. In the present work, SA-5K was purified from BCG CFs by affinity chromatography. A protein that could be detected in Western blot but not by standard protein staining techniques was obtained. When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M. tuberculosis. The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage. The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions. After size exclusion chromatography, reactivity-with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions. The latter MM was close to that estimated from the ORF of M. tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-BK released after reduction of a disulphide bridge. The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M. tuberculosis. The gene coding for SA-5K was cloned by PCR from BCG and M. tuberculosis DNA and was expressed in Escherichia coli.